Many biological processes in living cells are controlled by alterations in the levels or states of certain key proteins and metabolites. Measuring the levels of the various proteins that affect (or are affected by) the process is therefore important for gaining an understanding of the biological process. For example, a given hormone may, through a signaling cascade, activate certain key transcription factors which in turn induce the expression of a number of proteins with distinct activities. Comparison of the levels of the proteins in the cell prior to and after induction can indicate which gene products are being up regulated and/or down regulated by the action of the hormone. As a second example, comparison of the total complement of proteins from an organism (the proteome) prior to and after infection with a pathogen can show which proteins are up and/or down regulated by the infection. Such an analysis can provide important information about the mechanism by which the pathogen subverts its host cell, thereby aiding in the development of therapeutic drug strategies. Similarly, comparison of some or all of the proteins of the proteome before and after treatment with a drug can indicate the mechanism of action of the drug, as well as its potential effectiveness and toxicity. Yet another example, measurement of the state of phosphorylation of protein members of an intracellular cascade involved in turning on and off a given biological process can provide information about the control of the signaling pathway.
A facile method for accurately comparing the levels of proteins and other cellular components and biological materials as a function of time or as the result of particular treatment, such as a hormone, a drug, or a virus, as mentioned above, or an environmental stimulus, such as a temperature change, is needed. It is also necessary to assay these protein levels with high degree of accuracy because small changes in the levels of certain key proteins may, through a complex cascade of molecular events, produce large changes in the biological system.
Current proteomic analysis methods used different means of separations to profile proteins with different properties for comparison. A two dimensional gel can be used to separate proteins by their isoelectric points and sizes so that many proteins can be examined. High degree of reproducibility between samples is required to make meaningful comparison thus evolve multiplexing co-analysis methods. Proteins can be labeled with different fluorescent dyes such as Cy2, Cy3, or Cy5 and then combined together to be co-separate on the same 2-D gel simultaneously. The dyes are engineered so that their contribution in mass and charge to the proteins they label are the same or almost the same while having different absorption and emission spectra. As a result, after analysis the quantity of proteins originated from one sample can be determined and compared with other samples. Overall, enabling coding of samples so they can be combined for analysis and then decode at the end for quantitative comparison yield much more reliable analysis than separate side-by-side or sequential analyses of one sample at a time.
Another recent advance uses heavy isotope to label a protein sample to be combined with a non-labeled (normal isotope labeled) protein sample. The labeling can be done by heavy isotopes incorporation if the protein is undergoing synthesis by 15N, 13C, 18O . . . etc. There are also labeling reagents that label proteins that are already synthesized for analysis. One such reagent is called Isotope-Coded Affinity Tag (ICAT) reagent described in Aebersold et al. WO01/94935; WO03/102220; US2002/0168644; U.S. Pat. No. 6,670,194; WO03/102018; WO00/11208. This type of reagent not only allows labeling of proteins through specific functional groups such as amines, thio . . . etc. but also allows affinity selection of labeled proteins away from unlabeled molecules. A pair of ICAT reagent differs only in that one carry heavy isotope and the other carry normal isotope.
Similar to isotope labeling, using ICAT reagent to label two samples of proteins, one can then combine the two samples for co-analysis by any means that separate the different proteins apart so that they can be quantified and characterized individually. The interest here is to compare the quantity or level of abundance, thus mass spectrometry is used to make the determination. Upon analysis by mass spectrometry, proteins or peptides originated from different samples can be distinguished by their shifted mass peak. Then the peak height of one can be compared against that of the other thus enable quantitative comparison.
The majority of proteins don't increase or decrease in their expression levels due to a stimulus. The above analysis requires that mass spectrometry be used to analyze all proteins in both samples to spot just a few that are differentially abundant. Despite recent advances in instrumentation and supported software, mass spectrometry and especially analyses of the mass spectra themselves is still a very expensive and time consuming process. If just 1% of proteins between two samples are differentially abundant, then 99% of mass spectrometry analyses are redundant. As a result, there is a need for more efficient methods to quantitatively compare proteins between samples.